The objective of this proposal is to understand the critical biochemical steps in the mechanisms of transfection and transfection-enhancement. The enhancement of transfection in Bacillus subtilis by either phage infection or ultraviolet light irradiation of cells bears similairities to the SOS repair process. Evidence indicates that one basic aspect of the enhancement process the suppressson of exonuclease activity directed against single stranded DNA (xos) in enhanced cells. In phage enhanced cells, this goal is accomplished by the suppression of a specific Mg ions and Ca ions activated endonuclease. I have shown that one ntr mutant of Bacillus subtilis lacks most xos nuclease activity and that this mutant is completely lacking in transfection enhancement. DNA binding studies reveal that its efficiency (transfectants/micrograms DNA irreversibly absorbed) has been increased about thirty in phage enhanced cells. The mutant is constituitively enhanced. It is my intention to: 1. isolate and characterize the specific nuclease in this and other ntr minus mutants; 2. carry out genetic experiments to determine whether the phenotype of this xos mutant is dependent on only the absence of this specific xos nuclease activity or whether, as I suspect, another alteration which alone leads to quite different transfection and efficiency behavior must be present for the phenotype; and 3. undertake experiments to determine to what extent the enhancement process bears other similarities to the SOS repair process. In addition, I propose to continue analysis of 47 independently isolated ntr ion mutants of B. subtilis. These mutants demonstrate a wide variety of binding, entry, and other defects including at least 10 discernable mutants which have changes in nuclease activities. These mutants will be examined to decipher the roles of various nuclease and other enzyme defects in the transfection process.